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1.
Clin Cancer Res ; 26(23): 6284-6298, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32817076

ABSTRACT

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.


Subject(s)
Colorectal Neoplasms/prevention & control , Interleukin-12/administration & dosage , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/prevention & control , RNA, Messenger/administration & dosage , Th1 Cells/immunology , Tumor Microenvironment/immunology , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm , Female , Humans , Interleukin-12/genetics , Melanoma/genetics , Melanoma/immunology , Melanoma/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , RNA, Messenger/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Clin Cancer Res ; 23(13): 3416-3427, 2017 07 01.
Article in English | MEDLINE | ID: mdl-28069723

ABSTRACT

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.


Subject(s)
Glucocorticoid-Induced TNFR-Related Protein/immunology , Melanoma, Experimental/immunology , Oncogene Proteins, Fusion/administration & dosage , Tumor Necrosis Factors/immunology , Animals , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/immunology , Disease Models, Animal , Glucocorticoid-Induced TNFR-Related Protein/administration & dosage , Humans , Melanoma, Experimental/genetics , Melanoma, Experimental/therapy , Membrane Glycoproteins/agonists , Membrane Glycoproteins/immunology , Mice , OX40 Ligand , Oncogene Proteins, Fusion/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Tumor Necrosis Factors/agonists , Tumor Necrosis Factors/genetics
3.
Cancer Res ; 75(15): 3098-107, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-26081809

ABSTRACT

The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke.


Subject(s)
Interleukin-6/metabolism , Interleukin-6/pharmacology , Neovascularization, Pathologic/metabolism , Animals , Aorta/drug effects , Aorta/pathology , Calcium-Binding Proteins/genetics , Cell Line, Tumor , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Humans , Intercellular Signaling Peptides and Proteins/genetics , Interleukin-6/genetics , Jagged-1 Protein , Male , Membrane Proteins/genetics , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Neovascularization, Pathologic/chemically induced , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Organ Culture Techniques , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Pericytes/drug effects , Pericytes/pathology , Rats, Wistar , Serrate-Jagged Proteins , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Vesicular Transport Proteins/genetics , Xenograft Model Antitumor Assays
4.
FASEB J ; 27(10): 4244-53, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23825230

ABSTRACT

Junctional adhesion molecule C (JAM-C) is a transmembrane protein with significant roles in regulation of endothelial cell (EC) functions, including immune cell recruitment and angiogenesis. As these responses are important in promoting tumor growth, the role of EC JAM-C in tumor development was investigated using the ID8 syngeneic model of ovarian cancer. Within 10-15 wk, intraperitoneally injected ID8 cells form multiple tumor deposits and ascites that resemble human high-grade serous ovarian cancer. Compared to wild-type mice, survival in this model was increased in EC JAM-C knockouts (KOs; 88 vs. 96 d, P=0.04) and reduced in EC JAM-C transgenics (88 vs. 78.5 d, P=0.03), mice deficient in or overexpressing EC JAM-C, respectively. While tumor growth was significantly reduced in EC JAM-C KOs (87% inhibition at 10 wk, P<0.0005), this was not associated with alterations in tumor vessel density or immune cell infiltration. However, tumor microvessels from EC JAM-C-deficient mice exhibited reduced pericyte coverage and increased vascular leakage, suggesting a role for EC JAM-C in the development of functional tumor vessels. These findings provide evidence for a role for EC JAM-C in tumor growth and aggressiveness as well as recruitment of pericytes to newly formed blood vessels in a model of ovarian cancer.


Subject(s)
Cell Adhesion Molecules/metabolism , Gene Expression Regulation, Neoplastic/physiology , Immunoglobulins/metabolism , Ovarian Neoplasms/metabolism , Animals , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Female , Gene Expression Regulation/physiology , Humans , Immunoglobulins/genetics , Mice , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic , Ovarian Neoplasms/blood supply
5.
J Immunol ; 175(2): 1197-205, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-16002723

ABSTRACT

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-alpha- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-alpha-dependent activation of JNK and NF-kappaB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-kappaB activity exhibited enhanced invasiveness. Inhibition of the NF-kappaB pathway by TNF-alpha neutralizing Abs, an NF-kappaB inhibitor, RNAi to RelA, or overexpression of IkappaB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-kappaB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-kappaB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-alpha, via NF-kappaB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.


Subject(s)
Epithelial Cells/pathology , JNK Mitogen-Activated Protein Kinases/physiology , Macrophages/immunology , NF-kappa B/physiology , Neoplasm Invasiveness , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, CD/physiology , Basigin , Binding Sites/genetics , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Line, Transformed , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Female , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/biosynthesis , Macrophage Migration-Inhibitory Factors/physiology , Macrophages/enzymology , Macrophages/metabolism , Matrix Metalloproteinases/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Invasiveness/prevention & control , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , RNA Interference/physiology , Transcription Factor RelA , Up-Regulation/immunology
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